Abstract

Filarial nematode infections remain a significant threat of morbidity in the Tropics. Collectively known as filariasis, these helminth infections can result in gross inflammatory pathology of the infected host. However, more commonly, the host remains relatively asymptomatic, exhibiting a somewhat suppressed, non-inflammatory immune response to the parasite, permitting the longevity of infection commonly seen with many helminth infections. In addition to promoting parasite survival, such immunomodulatory actions appear conducive to host health by limiting development of pathological lesions resulting from aggressive, pro-inflammatory responses. This can prove to be advantageous for humans, as although nematode infection can result in severe pathology, the majority of infected people exhibit little evidence of an inflammatory response or overt tissue destruction/disruption commonly associated with autoimmune disorders. Consistent with this, there is increasing evidence supporting an inverse relationship between worm infection and Th1/17-based inflammatory disorders such as rheumatoid arthritis, inflammatory bowel disease, type-1 diabetes and multiple sclerosis. Moreover, in the developed world, there has been an alarming increase in these inflammatory diseases, coincident with recent improvements in hygiene; a trend not observed in parasite-endemic countries. Therefore, the ”hygiene hypothesis” proposes that these trends are directly associated, by predicting that in the absence of helminth infection, there is a lack of parasite-induced immunoregulation that can result in an over-active immune response in susceptible individuals, resulting in the development of autoimmunity.
Filarial nematodes are known to secrete immunomodulatory excretory-secretory products during infection, which act to modulate inflammatory host immune responses and thus, protect the parasite from elimination. ES-62, a phosphorylcholine-containing glycoprotein secreted by the rodent filarial nematode, Acanthocheilonema viteae, has previously been shown to modulate the responses of several innate cells to promote anti-inflammatory immune responses in vitro and in vivo. Indeed, this laboratory demonstrated that ES-62 exhibited anti-inflammatory properties that extended to potential therapeutic action in autoimmune inflammatory diseases such as Rheumatoid Arthritis (RA). This was evidenced by studies demonstrating that ES-62 not only reduced severity of disease in the murine model of collagen induced arthritis (CIA), but also acted to reduce pro-inflammatory cytokine and auto-antibody production from blood and synovial cultures derived from human RA patients. Perhaps rather surprisingly therefore, ES-62 was subsequently found not to offer any protection in some other models of inflammation, including the NOD mouse model of Type 1 Diabetes, the Plasmodium chabaudi model of malaria and the Toxoplasma gondii model of toxoplasma, all of which have been linked with Th1-like pathology. However, given the recent reassessment of CIA as a Th17-, rather than Th1-, mediated disease, it has been hypothesised that ES-62 exhibits therapeutic potential in models, such as CIA, which reflect Th17- rather than Th1 mediated pathology. As the previous CIA/RA studies did not address the role of the Th17 family of pro-inflammatory cytokine mediators, which have been implicated as orchestrating much of the pathology seen in CIA, the core aim of this thesis was to define whether ES-62 was mediating its therapeutic action in autoimmune inflammatory disorders by targeting this Th17 population. Indeed, in chapter 3 of this thesis, it was found that the significant inhibition of disease scores in ES-62-treated CIA mice was accompanied by a significant reduction in levels of IL-17-producing cells, as detected by flow cytometry. Moreover, such analysis revealed that ES-62 reduced IL-17 production from both CD4+ (Th17) cells and γδ cells, which were the two major IL-17-producing populations in the LN of CIA mice. By contrast, ES-62 was not found to modulate the levels of IFNγ-producing cells, either in terms of CD4 or CD8 T cells, which constitute the major cellular compartments generating this cytokine in CIA, supporting the hypothesis that ES-62 targets Th17/IL-17-, rather than Th1-associated inflammation.
In addition to suppressing CIA, pilot studies had shown that prophylactic treatment with ES-62 significantly inhibited the development of glomerulonephritis and articular inflammation in MRL/lpr mice, two pathologies commonly associated with SLE in humans. The anti-inflammatory actions of ES-62 were evidenced by a reduction of proteinuria levels and footpad swelling, respectively, but were not associated with modulation of lymphadenopathy, splenomegaly or hypergammaglobulinemia occurring in tandem with autoimmune pathology in such MRL/lpr mice. Studies in chapter 4 of this thesis characterised the cytokine profile of MRL/lpr lupus prone mice, in comparison to congenic MRL/MP control mice, throughout the course of disease induction and progression. These studies showed that IL-17 production was detected in the lymph nodes and serum of MRL/lpr mice, before the onset of lupus pathology. Furthermore, γδ T cells were the major IL-17 producing cell type examined in these mice. Prophylactic treatment with ES-62 resulted in reduced cytokine (IL-17 and IFNγ) release by lymph node cells and splenocytes in response to ex vivo stimulation with the mitogen, ConA, although this did not prove to be significant. As IL-17 was one of the cytokines targeted, this suggested that, as with the CIA model, ES-62 mediated its anti-inflammatory effects through modulation of this cytokine. Consistent with this, it was also found that exposure to ES-62 in vivo reduced the levels of IL-17-producing cells, particularly the γδ T cell population at the earliest time-point examined. By contrast, again as with the CIA model, there was no reduction in the levels of IFNγ-producing cells in ES-62 treated mice, confirming that ES-62 was not acting directly on these Th1 cells.
One of the hallmarks of the MRL/lpr model of lupus is the accumulation of B220+CD3+ Double Negative (DN) T cells, which account for much of the lymphadenopathy observed. These cells, which would otherwise have been deleted during development, are known to survive and accumulate due to the lpr (lymphoproliferative) mutation of the Fas gene in these mice. Despite being the predominant LN cell population following onset of disease in MRL/lpr mice, DN T cells have not generally been considered to be pathogenic in this model, but rather proposed to exhibit an anergic-like phenotype. However, some studies have suggested that they can produce cytokines, such as IFNγ and more recently it has been shown that they are a source of IL-17 [1, 2]. Moreover, DN T cells producing IL-17 have been found in the kidneys of SLE patients, suggesting they might indeed play a pathogenic role in lupus-like inflammation [3]. The data presented here established that such DN T cells are capable of producing a range of cytokines, including IL-10, IL-17 and IFNγ and indeed, the most novel finding of these studies was that DN T cells are capable of producing IL-22, a cytokine generally associated with Th17 cells. While the function of this IL-22 production is unclear, it was seen that the percentage of DN T cells producing IL-22 were significantly inhibited by exposure to ES-62 in vivo and hence, whilst ES-62 did not appear to influence the expansion of these cells, it did modulate their effector function by suppressing their capacity to produce this cytokine, although the absolute numbers of cells was not significant reduced.
In summary, the novel findings presented in this thesis support the theory that parasite-derived products such as ES-62 may protect against development of the autoimmune inflammatory diseases prevalent in developed society. The highly evolved anti-inflammatory mechanisms designed to beneficially maintain the host-parasite relationship could therefore potentially be exploited to design novel safe therapies for numerous inflammatory diseases. The precise mechanisms by which ES-62 mediates its anti-inflammatory effects have yet to be fully delineated, but it appears to target a variety of cells including IL-17- and IL-22-producing cells (γδ T cells, DN T cells and Th17 cells) in the inflammatory models examined. Importantly, the pilot studies testing the use of synthetic derivatives of ES-62, which showed promise with respect to modulation of inflammatory cytokine production from RA and SLE patient samples, highlight the potential for translating these results into a clinical setting.